Effect of Shiga Toxin and Its Subunits on Cytokine Induction in Different Cell Lines

Shiga toxins (Stxs) are bacterial virulence factors produced by Shigella dysenteriae serotype 1 and Escherichia coli strains. Stxs are critical factors for the development of diseases such as severe bloody diarrhea and hemolytic uremic syndrome. Additionally, Stxs trigger the secretion of pro- inflammatory cytokines and chemokines, particularly in monocytes or macrophages. The inflammatory cytokines result in the modulation of the immune system, local inflammations and enhancement of cytotoxicity. In this study, stimulation of the pro- inflammatory cytokines IL-1α, IL-1β, IL-6, IL-8, and TNF-α was assessed by recombinant Stx (rStx) and its subunits (rStxA and rStxB). Cytokines expression at mRNA level was investigated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) method in HeLa cells and THP1 monocyte/ macrophage cell lines. After incubation with rStx and its recombinant subunits, the expression of IL-1α, IL- 6 and IL- 8 mRNAs was strongly induced in HeLa cells. In HeLa cells, low expression of IL-1α mRNA was shown by rStxB induction. Furthermore, the expression of IL-1α and IL-1β mRNAs in undifferentiated THP1 cells was only induced by rStx. In differentiated THP1 cells, rStx and its recombinant subunits elicited the expression of IL-1α, IL-1β, IL-8 and IL- 6 mRNAs. On the other hand, expression of TNF-α mRNA was only induced by rStx. Based on the data, the profile of cytokine induction in response to the rStx, and its subunits differs depending on the cell types.

higa toxin (Stx) is a virulence factor produced by Enterohemorrhagic E. coli (EHEC) (1). It is also known as Verotoxin (VT) due to Vero cells' sensitivity to Stxs (2). Stxs are essential for the development of severe bloody diarrhea and hemolytic-uremic syndrome (HUS) with worldwide prevalence, characterized by serious complications including renal failure, thrombocytopenia and hemolytic anemia (3). Stxs have AB5 molecular structure, composed of one enzymatically active A subunit (32 kDa) and five identical B subunits (7.7 kDa each) that mediate the attachment of holotoxin (AB5) to neutral glycolipid receptor globotriaosylceramide (Gb3, or CD77) on the surface of host cells (4,5). StxA and StxB subunits are separately secreted into the bacterial periplasm where they are noncovalently assembled into holotoxin (6). The A subunit with highly S Submmited 4 March 2014; Accepted 22 April 2014; Published 20 May 2014 specific RNA N-glycosidase activity cleaves an adenine base of 28S ribosomal RNA (rRNA) of eukaryotic ribosomes (7). This is followed by the internalization of Stx into the target cell by endocytosis and transportation via the trans-Golgi network and Golgi apparatus to the endoplasmic reticulum (ER) and finally into the cytoplasm (8).
Thus, elongation-factor-1-dependent aminoacyl tRNA binding is blocked by depurination reaction, thereby rendering ribosomes inactive for protein synthesis which leads to cell death in susceptible host cells (9).
Despite the fact that Stx has direct effect on protein synthesis (also termed the ribotoxic stress response) (9), its function on exacerbating cytotoxicity is not only restricted to ribotoxic stress.
Stx also triggers programmed cell death (apoptosis) through various mechanisms in different cell types (10). It is crucial for the incidence of vascular lesions and tissue damage and subsequently translocation of the toxin into the bloodstream (11). Gb3 (14). Resistant cells generate soluble cytokines and chemokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). These are essential agents contributing to the susceptibility of endothelial cells (as the primary target) to toxin. This is mediated by increasing the expression of Gb3 and different leukocyte adhesion molecules to elicit cytotoxicity of the toxin (15). The host response factors play an essential role in the development of hemorrhagic colitis and HUS by inducing vascular damage (16).
Cell culture studies have shown that epithelial cell lines like Caco-2 and HCT-8 as well as human peripheral blood monocytes, primary human monocytes and macrophage-like cell lines can produce proinflammatory cytokines, especially the neutrophil chemoattractant IL-8 in response to sublethal or nontoxic dose of Stxs in vitro (17,18). On the other hand, it has been suggested that cytokine and chemokine expression is the result of Stx internalization inside the Gb3-negative cells (19).
However, retrograde transport pathway from Golgi to endoplasmic reticulum (ER) is necessary for Stx cytotoxicity at least in some cell types (20,21).
Thus eukaryotic cell lines with different Stx sensitivity (high and low content of Gb3) could synthesize pro-inflammatory cytokines.
In the present study, the expression of proinflammatory cytokines IL-1α, IL-1β, IL-6, IL-8, and TNF-α was assessed by treatment with rStx, rStxA and rStxB subunits in HeLa and THP1 monocyte/macrophage cell lines.

Holotoxin and its subunits
Stx as holotoxin and its subunits were obtained from previous study (22).The obtained crude toxins were applied to Endotrap (profos AG) (endotoxin removal systems in order to remove LPS content). The samples were sterilized by filtering through low protein binding filter (Miller®-GV 0.22 µm filter unit, Millipore) and the protein concentration was estimated using Bradford method by measuring absorbance at 595 nm with bovine serum albumin as control (Protein assay Kit, Bio-Rad).

Cell culture
The human myelogenous leukemia cell line THP-1 and cervical cancer HeLa cells were purchased from (National Cell Bank, Pasteur Institute of Iran) and cultured in RPMI-1640 medium (Biosera) supplemented with penicillin (100U/ml), streptomycin (100µg/ml ) and 10% fetal bovine serum. Cells were grown and maintained at 37°C in 5% CO2 in a humidified incubator.

Macrophage differentiation
The mature macrophage-like cells were

Detection of pro-inflammatory cytokine mRNA by reverse transcription-PCR (RT-PCR)
Total extracted RNA was amplified by one step AccuPower® RT-PCR Premix (BioNEER) Kit. All components necessary for cDNA synthesis and amplification were provided in one tube. The primers used in this survey are shown in Table 1. with the same amount of GAPDH RT-PCR product in order to obtain the same sensitivity for each cytokine. The extracted RNA and the reverse primer were mixed and incubated at 70ºC for 5 min.
The incubated mixture and the forward primer were then transferred to a premix tube. The cDNA synthesis was performed at 42ºC for 60 min and at 94ºC for 5 min. PCR cycles were carried out as follows: 94ºC for 60 sec, 54ºC for 30 sec and 72ºC for 60 sec with a final 10 min extension at 72ºC.
For all of the genes tested in this study (including the ubiquitous housekeeping gene GAPDH), 30 cycles of PCR was found optimal. The aliquots of PCR products hence obtained were visualized after electrophoresis on 2% agarose gel under UV in a gel documentation system and quantified by Lab Gel Doc Image software.

Statistical Analysis
The results were expressed as the Mean ±SEM for the number of experiments. The statistical significance was compared between each treatment group and the control by Student's t test. The analyses were performed using SPSS software version 18.

Cytokine mRNAs expression in HeLa cells
HeLa cells were treated with sub-lethal concentration of rStx, rStxA and rStxB subunits for 24 hours and then total cellular RNAs were extracted from treated and untreated cells and the expression of inflammatory cytokine mRNAs IL-6, IL-8, IL-1α, IL-1β, and TNF-α was examined by RT-PCR ( Figure 1A). In Figure 1B

THP1 cells
Optimal stimulation of cytokine expression by

Conflicts of interest
The authors declared no conflict of interest.